E2F1 regulates testicular descent and controls spermatogenesis by influencing WNT4 signaling

Abstract

Cryptorchidism is the most common urologic birth defect in men and is a predisposing factor of male infertility and testicular cancer, yet the etiology remains largely unknown. E2F1 microdeletions and microduplications contribute to cryptorchidism, infertility and testicular tumors. Although E2f1 deletion or overexpression in mice causes spermatogenic failure, the mechanism by which E2f1 influences testicular function is unknown. This investigation revealed that E2f1-null mice develop cryptorchidism with severe gubernacular defects and progressive loss of germ cells resulting in infertility and, in rare cases, testicular tumors. It was hypothesized that germ cell depletion resulted from an increase in WNT4 levels. To test this hypothesis, the phenotype of a double-null mouse model lacking both Wnt4 and E2f1 in germ cells was analyzed. Double-null mice are fertile. This finding indicates that germ cell maintenance is dependent on E2f1 repression of Wnt4, supporting a role for Wnt4 in germ cell survival. In the future, modulation of WNT4 expression in men with cryptorchidism and spermatogenic failure due to E2F1 copy number variations may provide a novel approach to improve their spermatogenesis and perhaps their fertility potential after orchidopexy.

Keywords: Copy number variations; Cryptorchidism; E2f1; Infertility; Testicular cancer; Wnt4.

Fig. 1.

E2F1 plays an important role in testicular descent. (A) WT mouse with normal testicular (T) descent (blue circles) into the scrotum below the preputial glands (PG). Inset shows the dissected testis. (B) Cremasteric sac of WT mice stained with Masson's trichrome indicating muscle (red) and collagen (blue) content. (C) TEM photomicrograph of the cremasteric sac of a WT mice, indicating normal organelles, including mitochondria (blue arrow), endoplasmic reticulum (black arrow) and nucleus (N). (D) E2f1-null mouse with left undescended testes (blue circles) beside the bladder (B, red circle). Right testis is in the normal position. Inset shows the dissected testis. (E) Cremasteric sac of an E2f1-null mouse with reduced muscle (red) and collagen (blue) content. (F,G) TEM of E2f1-null mice (n=6) in which 100% of the mice showed abnormal mitochondria (red arrows), indicated by the degenerate mitochondria with only their outer membranes visible, and with disorganized cristae and deteriorated mitochondria with concentric membrane rings inside a lysosome. (G) Autophagosomes were observed in 50% of the mice (inset). (H) Enlarged and dilated endoplasmic reticula (black arrow) were observed in 83% of the mice. (I) Mast cells (green arrow) were found in 67% of the mice. Magnification is indicated in the photomicrographs.

Fig. 2.

E2f1-null mice exhibit progressive infertility, testicular atrophy and decreased concentration of epididymal sperm. (A-K) Histological comparison using PAS staining of WT mice (A-C) displaying normal seminiferous tubule morphology and distribution, and testes of E2f1-null mice (E-G) displaying abnormal ST morphology at different postnatal days. (A) WT mice at P21. (B) WT mice at P90. (C) WT mice at P210. (D) Epididymal duct showing abundant spermatozoa in the lumen of WT mice. (E) E2f1-null mice at P21 had 79% normal seminiferous tubules, 18% abnormal seminiferous tubules (red asterisks) and 3% SCO tubules (green asterisks). (F) E2f1-null mice at P90 had 66% normal seminiferous tubules, 27% abnormal seminiferous tubules and 7% SCO seminiferous tubules. (G) Seminiferous tubules of E2f1-null mice at P210 were almost completely depleted of germ cells with 17% normal, 23% abnormal and 60% SCO seminiferous tubules. In addition, the testes show Leydig cell hyperplasia. (H) Spermatozoa were absent in the lumen of the majority of the epididymal ducts of the E2f1-null mice. (I) Fertility of WT (blue lines; n=6) was compared with E2f1-null mice (red lines; n=10) over 6 months. All WT mice produced six litters. One E2f1-null mouse was infertile and the remaining nine E2f1-null mice produced at least two litters before becoming infertile. (J) E2f1-null mice at any age had a significant decrease in the tubular diameter and epithelial thickness (n=5 in each group). (K) Quantification of the abnormalities in the seminiferous tubules of WT and E2f1-null mice in each group (n=5). For the WT group, the one with the highest percentage of abnormal tubules is represented (7 months of age). All magnifications are indicated. P-values are indicated. A Mann–Whitney test was used to determine significant difference in parameter between WT and E2f1-null mouse (*P<0.01). Scale bars: 50 μm.

Fig. 3.

E2f1-deficiency affects the expression of several genes encoding proteins functioning in signal transduction pathways. (A-C) Graphs represent the relative quantification (RQ) difference in gene expression between WT and null mice at 3 months of age in genes involved in cell cycle (A), apoptosis (B) and the Wnt-signaling pathway (C). The largest changes in testicular gene expression levels, stratified by age (at 3 weeks, 3 months and 7 months) for Serpina3n, Timp1 and Wnt4, are indicated (D). Data are mean±s.d. Statistical analysis was performed using REST (*P<0.01).

Fig. 4.

BTB and the junctional assembly is disrupted in E2f1^−/− mice. (A-C) In WT mice, the testicular injected biotin tracer (green) was present in the interstitial spaces and basal compartment but excluded from the adluminal compartment (nuclear counterstain indicated in blue, n=6). (D) Testis of mice injected with PBS had no testicular staining. (E-G) In 3-month-old E2f1^−/− testis, the biotin tracer passed through the BTB and entered the adluminal compartment in 60% of the tubules n=6; however, there are some normal seminiferous tubules in which biotin is excluded from the adluminal compartment (indicated by the asterisk). (H) Determination of the number of seminiferous tubules contained in the lumen biotin tracer in WT and E2f1^−/− mice at P90. There was a significant difference between WT and E2f1^−/− mice in seminiferous tubules containing biotin and the ones without biotin. Data are mean±s.d. P-values are indicated. A Mann–Whitney test was used to determine significant difference in parameter between WT and E2f1-null mouse (*P<0.01).

Fig. 5.

E2f1^−/− mice exhibited decreased numbers of undifferentiated spermatogonia. (A-H) Comparison of dual immunohistochemical staining with ZBTB16 (green), a marker of undifferentiated spermatogonia and SOX9 (red), a marker of Sertoli cells in testes of WT mice n=4 (A-D) and E2f1^−/− mice (E-H) n=5 at different postnatal days. (A) WT mice at P7. (B) WT mice at P14. (C) WT mice at P21. (D) Adult WT mice at P90. (E) E2f1^−/− mice at P7. (F) E2f1^−/− mice at P14. (G) E2f1^−/− mice at P21. (H) Adult E2f1^−/− mice at P90. (I) Quantification (mean±s.d.) of SOX9 cells at P7, P14, P21 and P90 indicated no significant difference between WT and E2f1^−/− mice. However, a significance difference in the number of ZBTB16 cells at P90 was observed. (J) Quantification of the number of seminiferous tubules (ST) containing ZBTB16⁺ cells in WT and E2f1^−/− mice at P90 indicated that only 16% of seminiferous tubules in WT mice did not contain ZBTB16⁺ cells compared with the 53% in the E2f1^−/− mice. A minimum of 25 seminiferous tubules were counted per mouse. Data are mean±s.d. A Mann–Whitney test was used to determine significant difference in parameter between WT and E2f1-null mice. *P<0.001.

Fig. 6.

Immunolocalization of WNT4 in mouse testes from birth to adulthood. WNT4 expression (DAB, brown staining) in WT mice at different postnatal days. (A) At P1, WNT4 was expressed in gonocytes (black arrow), peritubular (red arrow) and interstitial (green arrow) cells. (B) At P5, WNT4 was still expressed in gonocytes (black arrow), peritubular (green arrow) and interstitial (red arrow) cells. (C) CTNNB1 was expressed in WT mice at the basal compartment of the tubules and at Sertoli-germ cell junctions. (D) E2f1-null mice had a decrease in CTNNB1 expression, with few cells showing staining. (E,J-L) At P7, P14, P21 and P210, WNT4 was expressed in peritubular and interstitial cells. (F) At P14, WNT4 was expressed in interstitial cells. (G) At P21, WNT4 was expressed in interstitial cells. (H) At P90, WNT4 was expressed in interstitial cells. (I-L) WNT4 expression in E2f1-null mice at different postnatal days. (I) At P7, WNT4 was expressed in peritubular and interstitial cells. All sections were counterstained with Harris hematoxylin.

Fig. 7.

E2f1^−/−;Wnt4^f/f;Stra8^cre/+ mice exhibit a decrease in WNT4 testicular expression compared with E2f1^−/− mice. (A-D) WNT4 expression (DAB, brown staining) in the testes of 3-month-old mice (n=4). (A) WT mice exhibit almost no WNT4 expression. (B) In E2f1-null mice, WNT4 was present at the membrane and in the cytoplasm of spermatogonia and spermatocytes (black arrows). (C) In E2f1^−/−;Wnt4^f/f;Stra8^cre/+ mice, WNT4 staining is absent, except the non-specific sperm tail staining. (D) In Wnt4^f/f;Stra8^cre/+ mice, WNT4 immunohistochemical staining is absent. (E-H) CCND1 expression (DAB, brown staining) in the testes of 3-month-old mice (n=4). CCND1 was expressed in all mice analyzed. (I-L) Blue arrows point to CCND1 expressing spermatogonia. ZBTB16 expression (DAB, brown staining) in the testes of 3-month-old mice (n=4). ZBTB16 was expressed in all mice analyzed. Green arrows point to ZBTB16 expressing spermatogonia. (M) Quantification (mean±s.d.) of CCND1 cells indicated a significant difference in the number of CCND1 cells at P90 in E2f1^−/− mice compared with WT and E2f1^−/−;Wnt4^f/f;Stra8^cre/+ mice. (N) Quantification (mean±s.d.) of ZBTB16 cells indicated a significant difference in the number of ZBTB16 cells at P90 in E2f1^−/− mice compared with WT and E2f1^−/−;Wnt4^f/f;Stra8^cre/+ mice. All sections were counterstained with Harris hematoxylin at the designated magnifications. Data are mean±s.d. A Mann–Whitney test was used to determine significant difference in parameter between WT and E2f1-null mice (*P<0.01). ST, seminiferous tubules.

Fig. 8.

Wnt4-loss in germ cells in E2f1-null mice (E2f1^−/−;Wnt4^f/f;Stra8^cre/+ mice) improves spermatogenesis in E2f1-null mice. (A) Comparison of testis size of different genotypes in which E2f1^−/− and E2f1^−/−;Wnt4^f/f are smaller than E2f1^−/−;Wnt4^f/f;Stra8^cre/+ and Wnt4^f/f;Stra8^cre/+. The E2f1^−/−;Wnt4^f/f;Stra8^cre/+ testis is highly vascularized similar to WT (not shown). (B) Testicular histology of E2f1-null mice. (C) Testicular histology of E2f1^−/−;Wnt4^f/f;Stra8^cre/+ mice. (D-K) Comparison among WT mice (blue), E2f1-null mice (red) and E2f1^−/−;Wnt4^f/f;Stra8^cre/+ (green) of their fertility (D), average number of pups per litter (E), testicular weight (F), epididymal weight (G), sperm count (H), sperm motility (I), and gene expression patterns (J,K). A Mann–Whitney test was used to determine significant difference in parameter between WT and E2f1-null mice (*P<0.01). Data are mean±s.d. Scale bar: 50 μm.

Fig. 9.

Model for the function of E2f1 as a master regulator with important roles in testicular descent and germ cell survival. E2f1 has two important roles in testicular function. First, E2f1 plays a role in the inguinoscrotal phase of testicular descent as E2f1-null mice have inguinal cryptorchidism. Second, E2f1 plays a role in germ cell maintenance by repressing Wnt4 in spermatocytes. E2f1-null mice with spermatogenic failure express WNT4 in germ cells. In addition, under pathological conditions, WNT4/CTNNB1 signaling can be activated in Sertoli cells, allowing the expression of WNT4 inside the seminiferous tubule, leading to germ cell loss (Boyer et al., 2012). WNT4 repression is important for germ cell survival as in the absence of E2f1, germ cells are depleted, but after Wnt4 expression is ablated in spermatocytes fertility is restored. However, beside repressing Wnt4, E2f1 has additional testicular function that probably involves the regulation of genes important for germ cell proliferation and differentiation, such as Ccnd1 and Gdnf as E2f1^−/−;Wnt4^f/f;Stra8^cre/+ mice have significantly smaller testes compared with WT mice.